33signals Fgf8 and Wnt1. The self-formation of isthmic organizer tissue in this culture
conditions is also dependent on Fgf8 and Wnt1 and suppressed by their inhibitors
(Muguruma et al. 2010 ), suggesting that an autoregulatory feedback loop operates
in the maintenance of this tissue in small area. Direct application of Fgf8 and Wnt1
might destabilize this autoregulatory loop (Sasai 2013 ).
This self-formation principle is applicable to the generation of human cerebellar
neurons from hPSC with some minor modification. This includes the addition of
TGFß inhibitor, which inhibits mesenchymal differentiation and promotes neuroec-
todermal differentiation (Muguruma et al. 2015 ). These hPSC-derived neural pro-
genitors express En2 and Gbx2, midbrain-hindbrain markers, after 21 days in
culture. Around 35 days in culture, substantial populations of hPSC-derived cells
express Purkinje cell progenitor markers, Lhx5, Kirrel2, Ptf1a, and Skor2. Following
long-term coculture with mouse RL-derived granule cells, hPSC-derived cerebellar
progenitors become to L7+, Calbindin+, GABA+, and Aldolase C+ (specific markers
for mature Purkinje cells). L7+/Calbindin+ hPSC-derived Purkinje cells developed
elaborate dendritic branches and spines that are positive for Purkinje cell-specific
glutamate receptor GRID2 (GluRδ2). GRID2 was associated with CBLN1, which
is expressed at the presynaptic termini of the parallel fibers. The electrophysiologi-
cal properties of hPSC-derived Purkinje cells showed typical characters as seen in
rodents (Muguruma et al. 2015 ) (Fig. 2.5).
It is known that BMP signals from the roof plate promote RL formation (Alder
et al. 1999 ). We previously showed that BMP signals are necessary for the induction
of RL-derived Atoh1+ progenitors in mESC culture (Muguruma et al. 2010 ). In con-
trast, Atoh1+ cells, Atoh1+/Barhl1+ granule cells, and Tbr1+/SMI-32+ DCN projec-
tion neurons were generated from hPSCs even in the absence of BMP4 (Muguruma
et al. 2015 ).
2.2.4 Self-Formation of Polarized Cerebellar Structure
The formation of polarized cerebellar neuroepithelium is more or less stochastic.
Interestingly, sequential application of Fgf19 and SDF1 (CXCL2) promoted self-
formation of polarized cerebellar tissue consisting of a RL-like region at the edge of
the sheet and the rest of the sheet displaying an identity reminiscent of the cerebellar
field. Remarkably, in this condition, hPSC-derived cerebellar tissue displays an api-
cobasally layered arrangement of multiple cell types, including progenitors similar
to the cerebellar VZ, an intermediate layer containing precursors of Purkinje neu-
rons, and an outermost layer occupied by derivatives of the RL, which have the
characteristics of granule cell precursors (Fig. 2.6). hPSC-derived neural progeni-
tors appear to have more potential self-organizing ability to differentiate into cere-
bellar tissues than those from mouse PSCs.
2 Self-Organized Cerebellar Tissue fromfiHuman Pluripotent Stem Cells andfiIts...